Enzymes are important proteins which catalyze chemical reactions in a living body. The localization and activities thereof are strictly controlled. It has been found that the localization of enzyme changes and abnormal increase or decrease of enzyme activity occurs in the tissues and cells affected by various diseases.
In tumor cells, the glycolysis using glucose as a raw material plays an important role in producing energy. Particularly in tumor cells present in a low-oxygen environment, the glycolysis acts as a major energy supply source. In the glycolysis, various enzymes act. For example, lactic dehydrogenase is the enzyme which acts in the final stage of the anaerobic glycolysis and converts pyruvic acid to lactic acid. Monitoring (metabolism mapping) the enzyme activity in the glycolysis is desired as a mean for enabling early detection of a solid tumor present in a low-oxygen environment.
A proteolytic enzyme, protease, is a general term used to refer to an enzyme catalyzing hydrolysis of a peptide bond. In a living body, a protease digests a protein into amino acids serving as a nutrient source and decomposes unnecessary proteins in cells. A protease has extremely large physiological significance.
Matrix metalloprotease (MMP) is an enzyme playing a major role in an abnormal site such as a disease site represented by a tumor. MMP catalyzes a decomposition process of extracellular matrix, which is required for local growth and metastasis/infiltration of a tumor (Deryugina E I et al., Cancer Metastasis Rev. v 25, 9-34. (2006)). The extracellular matrix consists of substances supporting cells in vivo, such as collagen, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminine and vitronectin. In a tumor site, expression of a plurality of types of MMPs is placed under temporal-spatial control. Most of them are known to be highly expressed. Also, MMP expression increases during metastasis/infiltration of a tumor. For these reasons, the MMP family has been expected to work as a tumor marker and an effective target in developing anticancer agents.
It is extremely important and efficient to monitor the activity of an enzyme such as MMP, which is closely associated with a disease, in the fields of medical/biological studies, clinical test and internal diagnostic imaging. To monitor enzyme activity with high sensitivity and high accuracy, it is necessary to develop probe molecules for monitoring enzyme activity with high sensitivity and a method for detecting the probes.
Probes to be used in-vivo and in-vitro for detecting the activities of enzymes such as a protease have been disclosed in documents. In a method generally employed, a substrate to a degrading enzyme is labeled with a chromophore or a luminophore. After the substrate is cleaved by the degrading enzyme, color or luminescence is generated, which is measured to detect enzyme activity. In the field of diagnostic imaging, reports have been made of a nuclear MRI (magnetic resonance imaging) probe using gadolinium for detecting β-galactosidase activity (e.g., Moats R A et al., Angew Chem. Int. Ed. Engl., v 36, 726-728 (1997)), a fluorescent probe using a near-infrared fluorescent dye for detecting protease activity (e.g., Japanese Patent Application Laid-Open No. 2002-514610 and Bremer C et al., Nature medicine, v 7, 743-748 (2001)) and an MRI probe using magnetic nano particles for detecting an enzyme activity (e.g., Perez J M et al., Nature Biotechnology, v 20, 816-820 (2002)).
These conventional techniques have the following problems that have remained unsolved. First, when an MRI probe is used, degree of relaxation caused by enzymatic activation is relatively low. As a result, it cannot be clearly distinguished that degree of relaxation is observed or that the concentration of probes is observed. When a luminescent probe or a fluorescent probe is used, it is difficult to capture a signal sent from deep part of a living body since permeability of light through the living body is low and light is scattered in the living body. Another problem resides in the specificity of a probe to a substrate, which decreases due to chemical modification and labeling. In addition, a probe has low biocompatibility. Consequently, the dose of the probe comes to be limited, with the result that a signal/noise ratio decreases when enzyme activity is monitored. A substrate probe and a method for detecting enzyme activity that solve these problems have not yet been found.
In the meantime, in order to determine the distribution and structure of a substance of interest in a living body with the lapse of time, NMR (nuclear magnetic resonance) detection using a stable isotope-labeled compound has been employed (see Japanese Patent Application Laid-Open No. 2000-290291 and Watanabe H et al., Magnetic Resonance in Medicine v 43, 525-533 (2000)). In Japanese Patent Application Laid-Open No. 2000-290291, an oligonucleotide labeled with 13C and 15N is detected in a body composition. In Watanabe H et al., Magnetic Resonance in Medicine v 43, 525-533 (2000), metabolism mapping of 13C glucose administered to a living body is made. However, up to present, detection of protease activity by a multi-dimensional (three dimensional or more) nuclear magnetic resonance method using a stable-isotope labeled substrate probe has not yet been made and a substrate probe to be used in the method has not been disclosed, either.